**5. Conclusions**

Each of the methods described in this study has its pros and cons when applied in the laboratory to characterize the gu<sup>t</sup> microbiota. Classical culturing is relatively cheap and widely available, but it has been shown that culture-based methods can only detect up to 20% of all the bacterial taxa present in a given sample. Methods based on next-generation sequencing are more precise, non-dependent on the cultivation step and can describe every bacterial community with more detail. However, they are also much more expensive, laborious, may introduce PCR biases and do not discriminate between live and dead microorganisms. To tackle this problem, methods such as flow cytometry with fluorochromes (FCM) can be used, as they enable discriminating between live and dead bacteria. Based on this study and the literature searches, it is clear that there is no universal method for the assessment of human gu<sup>t</sup> microbiota, and especially that some methods, such as FCM, may produce only limited results. However, when combined, the methods presented in this study may generate a detailed picture of the fecal microbiota solution for FMT.

In this study, we showed that fecal cultures are characterized by very high variability. By creating a Venn diagram, a "core cultivable microbiota" was identified, and it was composed of only 16 species. The largest number of species, mainly anaerobic, were obtained from donor C, who is a regular donor of stool for FMT. Donor C was characterized by a statistically significantly higher content of species considered beneficial (*Faecalibacterium*, *Bacteroides*, *Barnesiella*, *Blautia*, *Roseburia*, *Butyricimonas*). The general biodiversity of the stool microbiota of donor C was statistically significantly higher than from donors A and B. Microbiota stability over time was also transparent for donor C. In assessing the viability of bacterial cells, three groups of cells were distinguished: alive, dead and unknown. A population of "unknown" cells contained a group of double negative cells (SYTO9-, PI-). The presence of the double negative cell population correlates with the relative amount of *Anaeroplasma*, which appear more frequently in gu<sup>t</sup> microbiota of "non-donors" (donors unsuitable for FMT).

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2077-0383/9/7/2036/s1, Table S1: Table depicting raw values obtained from the flow cytometry experiment for all of the investigated samples; Table S2: Table depicting numbers of amplicon sequences remaining after each step of pre-analysis quality filtering.

**Author Contributions:** Conceptualization, J.B.; methodology, J.B., G.W.B., L.D., M.D., A.S.-E., E.P., T.D. and P.G.; formal analysis, M.D., L.D. and J.B.; investigation A.S.-E., E.P., J.B., L.D. and M.D.; writing—original draft preparation, J.B., M.D. and L.D.; writing—review and editing G.W.B., A.S.-E., E.P., T.D., and P.G.; funding acquisition, J.B. and G.W.B. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Incubator of Innovation + program financed by the Ministry of Science and Higher Education, Poland, and financed under EU funds (SG OP), gran<sup>t</sup> number 1WP/FS200/ZW2/17.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.
